Many medically significant biological processes are mediated by proteins participating in signal transduction pathways involving G-proteins and second messengers such as cAMP. The membrane protein gene superfamily of G-protein coupled receptors (GPCRs) includes a wide range of biologically active receptors, such as hormone, viral, growth factor, and neuro-receptors. GPCRs have been characterized as having seven putative transmembrane (TM) domains (designated TM1, TM2, TM3, TM4, TM5, TM6, and TM7), which are believed to represent transmembrane α-helices connected by extracellular or cytoplasmic loops. Most G-protein coupled receptors have single conserved cysteine residues in each of the first two extracellular loops that form disulfide bonds believed to stabilize the functional protein structure. G-protein coupled receptors may be coupled intracellularly with heterotrimeric G-proteins and various intracellular enzymes, ion channels, and transporters. Different G-protein α-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell.
One important subfamily of the GPCRs is the corticotropin-releasing factor receptors (CRFR), also known as corticotropin-releasing hormone receptors (CRHR). Corticotropin-releasing factor (CRF) (corticotropin-releasing hormone) is a 41-residue hypothalamic peptide (SEQ ID NO:5) which stimulates the secretion and biosynthesis of pituitary ACTH. Secretion of ACTH leads to increased adrenal glucocorticoid production. CRF was isolated and characterized based on its role in the hypothalamic-pituitary-adrenal axis (HPA). More recently, however, it has been found to be distributed broadly within the central nervous system (CNS), as well as in extra-neural tissues such as the adrenal glands and testes, where it may also act as a paracrine regulator or neurotransmitter.
A considerable body of evidence suggests that peptides of the CRF family, e.g., CRF(1), (frog) sauvagine, (fish) urotensin, and the mammalian urocortins 1, 2 and 3 (Ucn 1,2 and 3), play biologically diverse roles by activating CRF receptors. The CRF receptors, encoded by two distinct receptor genes, exist in multiple splice variant forms and display both species and tissue differential expression. Studies with transgenic mice expressing functionally disabled receptors, have underscored the importance of CRFR1 in regulating the hypothalamic-pituitary-adrenal axis in its response to stress. CRFR2 plays an important role in modulating the central nervous system response to stress as well as a unique role in cardiac function and pancreatic hormone release.
The CRF receptors belong to the B1 subfamily of G-protein coupled receptors (GPCRs). The GPCRs present a large variety of different conformations in their extracellular domains in order to cover their different functions. Indeed, a major ligand-binding site on both CRFR1 and CRFR2 is the N-terminal extracellular domain (ECD1). The inhibitory binding constants for a bacterially expressed soluble protein fragment, ECD1-CRFR2β, (comprising amino acids 39-133 of mCRFR2β) are: 11.8 (7.4-18.9) nM, 53.7 (18.7-154) nM and 21.1 (15.3-29.0) nM for Ucn 1, Ucn 2, and astressin, respectively. There remains a need for additional compositions and methods for identifying and assessing agonist and antagonist of B1 GPCRs, particular corticotropin releasing factor receptors.